The cholinergic hypothesis of rapid eye movement (REM) sleep generation proposes that the REM phase of the mammalian sleep cycle is generated, in part, by pontine cholinergic neurotransmission. The long term objective of this application is to identify the mechanisms by which hypocretin and gamma aminobutyric acid (GABA) modulate cholinergic neurotransmission in brain regions known to regulate sleep and wakefulness. The proposed experiments will use the techniques of in vitro autoradiography, in vivo microinjection and microdialysis, high performance liquid chromatography (HPLC), and polygraphic recordings of sleep and wakefulness to test hypotheses outlined in four specific aims. Aim 1 will test the hypothesis that some hypocretin receptors in arousal-related brain stem nuclei of rat couple to inhibitory guanine nucleotide binding (G) proteins. This hypothesis will be tested by blocking hypocretin-l-induced [35/S] guanylyl-5'-O-(gamma-thio)triphosphate ([35/S]GTPgammaS) binding and hypocretin-l-stimulated acetylcholine (ACh) release with pertussis toxin, a selective blocker of inhibitory G proteins. Aim 2 will use in vitro autoradiography to test the hypothesis that hypocretin-stimulated [35S] GTPgammaS binding in arousal-related brain stem nuclei is increased in transgenic mice that have a genetic ablation of hypocretin-containing neurons. Aim 3 will test the hypothesis that the REM sleep-like state produced by pontine reticular formation microinjection of the GABAA receptor antagonist bicuculline results from increased ACh release caused by removal of GABAergic inhibition. This hypothesis will be tested using cat and mouse. Aim 4 will focus on the basal forebrain, testing the hypothesis that GABA release within the substantia irmominata region of cat basal forebrain varies significantly across the sleep-wake cycle and is modulated by nitric oxide. The health relatedness of this project lies in the facts that most major psychiatric disorders are characterized by abnormal sleep, and common neurobiological mechanisms are thought to underlie REM sleep and certain forms of depression. These preclinical studies will provide basic information of potential relevance for psychiatry and sleep disorders medicine.